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It seems that yet another domain within the organism
touched by Ca
2+
and cAMP is the extracellular space. It
has long been known that intracellular signaling events
are associated with changes in second messenger con-
centrations outside the cell. Can these fluctuations be
regarded as “signals” in their own right? This article will
address how cAMP and Ca
2+
move across the cell mem-
brane, the potential mechanisms for sensing these
extracellular changes in messenger concentration, and
the physiological outcomes of these signaling events.
Export of cAMP from
Stimulated Cells
In 1963, just 5 years after the seminal descriptions of
the second messenger function of cAMP, Davoren and
Sutherland reported the existence of a probenecid-
sensitive mechanism for cAMP extrusion in nucleated
pigeon erythrocytes (18). Over the years, numerous
reports have appeared demonstrating that cAMP can
be expelled following agonist stimulation from a wide
variety of cell types, including adipocytes (57), hepato-
cytes, renal epithelial cells (56), neuronal cells (53),
fibroblasts, T lymphocytes (67), and skeletal muscle
(27). In some earlier studies, investigators even used
determinations of external [cAMP] as a surrogate for
measurements of hormone-dependent cAMP accu-
mulation inside the cell (8). Nevertheless, this ability
of cells to eject the second messenger is apparently
cell-type specific, since certain other cell types do not
appear to extrude cAMP whatsoever.
cAMP export can proceed across a concentration
gradient, is temperature dependent, and requires ener-
gy in the form of ATP (FIGURE 1). This process is sus-
ceptible to inhibitors of organic anion transport, such
as probenecid and sulfinpyrazone (56). In a colonic
carcinoma model (CC531
mdr+
), the efflux of cAMP was
found to be exquisitely sensitive to cell swelling (28).
Certain multi-drug resistance proteins in the MRP fam-
ily have now been identified as significant pathways for
cAMP egress (39, 69). These proteins belong to the
superfamily of ATP binding cassette (ABC) proteins,
specifically the ABCC subfamily. Most of these are
known to function as active membrane transporters for
organic anions and various drugs [e.g., for bile acids
and chemotherapeutic agents, including nucleoside
320
1548-9213/05 8.00 ©2007 Int. Union Physiol. Sci./Am. Physiol. Soc.
The foundations of the second messenger concept were
established nearly 50 years ago when Earl Sutherland
and Ted Rall identified a heat stable factor that mediat-
ed the intracellular actions of the hormones glucagon
and epinephrine on glycogen metabolism in the liver
(49). That factor was, of course, cyclic AMP, a discovery
that earned Sutherland a Nobel Prize in 1971 (58).
The next signaling molecule to be officially consid-
ered as a second messenger was the calcium ion (12).
The importance of Ca
2+
as a trigger of muscle contrac-
tion was known since the 19th century from the pio-
neering work of London-based physiologist Sydney
Ringer. However, acceptance of the general signal
transduction function of Ca
2+
, as originally proposed
by Lewis Victor Heilbrunn in the 1940s, initially met
with resistance (43). Recognition of the validity of this
theory slowly built following the identification of the
Ca
2+
ATPases, intracellular Ca
2+
release channels, and
protein targets for the Ca
2+
signal throughout the 1960s
and 1970s. Around this same time, several laboratories
managed to make direct measurements of agonist-
stimulated intracellular Ca
2+
transients using the lumi-
nescent jellyfish photoprotein aequorin. Together,
these findings firmly vaulted Ca
2+
to the status of a
bona fide second messenger.
Ca
2+
and cAMP are now recognized as universal reg-
ulators of cell function. Between them, these two clas-
sic textbook second messengers impact nearly every
aspect of cellular life in diverse organisms ranging
from amoebae to plants to human beings. A recurrent
theme that has emerged since the early descriptions of
these fundamental messengers is the importance of
localized signaling events within the cell (6, 16, 60, 70).
By confining Ca
2+
or cAMP to precise subcellular
domains (e.g., plasma membrane, organelles), these
molecules can selectively activate a subset of targets,
thereby expanding the repertoire and range of the sig-
nal. Discovery of this property was greatly facilitated
by the parallel development of fluorescent probes and
digital microscopic imaging techniques, methods ini-
tially applied for the visualization of Ca
2+
signaling
events (71). More recently, FRET-based indicators for
imaging cAMP in single cells have been used to con-
firm that the metabolism and disposition of cAMP can
be regulated independently in different parts of the
cell (25, 42, 46).
REVIEWS
Extracellular Calcium and cAMP: Second
Messengers as “Third Messengers”?
Aldebaran M. Hofer and
Konstantinos Lefkimmiatis
Department of Surgery, VA Boston Healthcare System and
Brigham & Women’s Hospital, Harvard Medical School,
West Roxbury, Massachusetts
ahofer@rics.bwh.harvard.edu
Calcium and cyclic AMP are familiar second messengers that typically become ele-
vated inside cells on activation of cell surface receptors. This article will explore
emerging evidence that transport of these signaling molecules across the plasma
membrane allows them to be recycled as “third messengers,” extending their abili-
ty to convey information in a domain outside the cell.
PPHHYYSSIIOOLLOOGGYY 2222:: 332200––332277,, 22000077;; 1100..11115522//pphhyyssiiooll..0000001199..22000077
analogs; see Kruh and Belinsky for review (39)]. In par-
ticular, MRP4, MRP5, and MRP8 are established trans-
porters of cyclic nucleotides (cAMP and cGMP) (69).
These drug efflux pumps often assume polarized distri-
butions in epithelial cells (e.g., MRP4 is found in the
apical membrane of kidney tubule cells and the baso-
lateral membrane of prostate glandular cells) (39).
Quantitatively, the amount of cAMP expelled by cer-
tain cell types can be quite significant. For instance,
human CD4
+
T lymphocytes stimulated with cholera
toxin release more than 50% of the total cAMP pro-
duced over a 24-h period to the extracellular space
(67). Strewler found an even more vigorous degree of
probenecid-sensitive export in polarized LLC-PK
1
renal epithelial cells; 40 min after stimulation with
arginine vasopressin, the external cAMP concentra-
tion in the apical bath was more than twice that
retained inside the cells (56). In humans, the concen-
tration of cAMP in plasma and urine can become dra-
matically elevated under a variety of physiological
conditions and also following infusion of exogenous
cAMP-elevating hormones (particularly epinephrine,
parathyroid hormone, and glucagon). For example,
Hendy and colleagues showed many years ago that
infusion of human subjects with a large bolus of
glucagon causes massive release of cAMP from the
liver, elevating plasma levels from low nanomolar lev-
els to over 450 nM within 10 min (31). This could
translate into dramatic local accumulations of cyclic
nucleotides in the diffusion-restricted spaces adjacent
to cells. Direct measurements using microdialysis
techniques have further demonstrated that significant
fluctuations in extracellular cAMP can occur in intact
brain tissue during agonist treatment (11).
The cAMP extrusion mechanism has been largely
dismissed as a means of regulating the intracellular
cAMP signal, since the cyclic nucleotide phosphodi-
esterases (PDEs: the enzymes that degrade cAMP and
cGMP) already provide powerful and rapid control of
intracellular cAMP (3, 39). Furthermore, it has been
argued that this strategy is too energetically unfavor-
able to be a viable means of regulating the cyclic
nucleotide levels within the cell, with the further
adverse consequence of depleting the cellular purine
pool (38). As seen in FIGURE 2, however, acute inhibi-
tion of this efflux pathway with probenecid can result in
measurable increases in resting intracellular [cAMP] as
measured using sensitive FRET-based reporters. This
suggests that in some cells this molecular device may
be capable of altering resting cAMP levels and, poten-
tially, the profile of the intracellular cAMP signal (27,
56), in addition to altering extracellular levels of cAMP.
Actions and Targets of
Extracellular cAMP
In the social amoeba Dictyostelium discoideum, extra-
cellular cAMP serves as a chemical alarm bell that
signals single-celled individuals to aggregate during
stress into a multicellular “slug” or pseudoplasmodi-
um. This fascinating organism, long used as a model
for cellular migration and chemotaxis, is known to
express four G-protein-coupled receptors (GPCRs)
used to detect secreted cAMP (cAR1, cAR2, cAR3,
cAR4; not to be confused with the Ca
2+
-sensing recep-
tor “CaR” described below) (5). These receptors have
affinities for cAMP ranging from sub-micromolar to
micromolar.
To date, no mammalian homologs of this well char-
acterized cAMP receptor have been reported. It is
noteworthy, however, that the Dictyostelium GPCRs
share some limited sequence similarity to the secretin
family of GPCRs expressed in mammalian cells,
which includes receptors for parathyroid hormone
and calcitonin. It has been speculated that the latter
class of GPCRs may mediate the actions of extracellu-
lar cAMP in vertebrates (5). Extracellular cAMP is
known to have many actions on diverse organ
321
PHYSIOLOGY • Volume 22 • October 2007 • www.physiologyonline.org
REVIEWS
FIGURE 1. “Third messenger” activity of extracellular cAMP
Intracellular cAMP is typically generated via adenylyl cyclases (AC) fol-
lowing hormonal activation of G-protein-coupled receptors (GPCRs)
linked to the heterotrimeric G-protein, G
s
. Once formed, the second
messenger can be actively transported to the extracellular space via a
probencid- and sulfinpyrazone-sensitive efflux mechanism belonging to
the ATP-binding cassette (ABC) transporter family. Extracellular cAMP is
hypothesized to have direct actions on putative receptor proteins (as of
yet unidentified) that are expressed on neighboring cells. Alternatively,
it is well established that extracellular cAMP can be sequentially metab-
olized, first by ecto-phosphodiesterase to adenosine monophosphate
(AMP), and then by ecto-5’-nucleotidase to adenosine. Adenosine can
then act as a paracrine or autocrine messenger to activate other signal
transduction cascades via one of four subtypes of adenosine receptors
(A
1
, A
2A
, A
2B
, A
3
). In addition, since cAMP is relatively stable in blood,
circulating cAMP can be converted by ecto-enzymes at a distant site,
effectively rendering cAMP as a prohormone for adenosine (which has a
fleeting half-life in the circulation).
cell model (22). Again, intracellular cAMP and extra-
cellular cAMP had opposing actions on PGHS-2
expression, causing upregulation and downregulation
of the enzyme, respectively.
Paracrine Action of cAMP on the
Renal System: The Extracellular
cAMP-Adenosine Pathway
The hormone glucagon, which is released from the
pancreas directly into the portal blood flow, causes
intracellular cAMP signaling in hepatocytes, and, as
suggested above, this leads to substantial efflux of
cAMP into the general circulation. It appears that an
important target of this circulating cAMP is the renal
system, particularly the proximal tubule. Glucagon is
well known to cause a marked enhancement of renal
sodium and phosphate excretion in vivo, although
specific receptors for glucagon have never been iden-
tified in the kidney. This prompted Bankir and
colleagues (5) and others to propose that cAMP
released from the liver might be acting as a
circulating factor mediating the renal actions of
glucagon. A sequence of studies by Ahloulay et al.
showed that cAMP infusion alone reproduced the
actions of glucagon on renal Na
+
and PO
4
–2
handling
(2). This phenomenon has been named the
“pancreato-hepatorenal cascade.”
A comprehensive series of animal experiments car-
ried out by Jackson and coworkers (36, 37) have given
a further twist on this general theme. These investiga-
tors showed that the cAMP entering the general
circulation from the liver is able to undergo enzymatic
conversion to adenosine once it reaches the kidney
(FIGURE 1). Adenosine has a short half-life in the cir-
culation (~1 s); therefore, cAMP (which is stable in
plasma) may be regarded as a sort of prohormone for
adenosine. Once produced (either locally or at a dis-
tant site), adenosine can activate one of four different
receptor subtypes (A
1
, A
2A
, A
2B
, and A
3
). Complex sce-
narios can be envisioned considering that adenosine
receptor subtypes A
1
and A
3
interact with G
i
/G
o
to
reduce intracellular cAMP levels in target cells, where-
as the A
2A
and A
2B
subtypes serve to increase cAMP via
G
s
. Therefore, cAMP released from one cell type could
conceivably initiate cAMP signaling in a neighboring
cell or suppress cAMP signaling depending on the par-
ticular adenosine receptor subtype expression pattern.
As described above, the cAMP-adenosine pathway
is prominent in the kidney, but substantial evidence
for this phenomenon also exists in the central nervous
system (11, 21, 38). Moreover, the cAMP-adenosine
pathway has been speculated to be important in the
cardiovascular system and also for systemic metabolic
homeostasis (57). The presence of the pathway does
not preclude the possibility that cAMP may exert
direct actions on cells in addition to indirect effects via
adenosine production.
322
PHYSIOLOGY • Volume 22 • October 2007 • www.physiologyonline.org
systems, including renal, hepatic, and central nervous
system. This subject has recently been reviewed in depth
by Bankir et al. (5) and Jackson and Raghvendra Dubey
(38). As described below, some of these actions may be
the indirect result of the metabolism of cAMP to adeno-
sine in the extracellular space (the “extracellular cAMP-
adenosine pathway”), although some effects appear to
be direct. For example, Sorbera and Morad (55) showed
in 1991 that 50 M extracellular cAMP rapidly (~50 ms)
inhibited a sodium current in ventricular myocytes
derived from several vertebrate species. This effect was
sensitive to pertussis toxin, suggesting a GPCR-based
mechanism dependent on G
i
or G
o
proteins (55). As
another example, secreted cAMP (but not adenosine)
derived from stimulated human CD4
+
T lymphocytes
was recently shown to exert significant growth effects on
neighboring T cells in a co-culture system (67).
Detrick and colleagues demonstrated that nanomo-
lar concentrations of extracellular cAMP and cGMP
(but not adenosine or guanosine) enhanced colony
formation in myeloid progenitor cells. Interestingly,
membrane permeant forms of the cyclic nucleotides
used at high micromolar concentrations had the
opposite effect on the proliferation of the cells, imply-
ing that an intracellular elevation of cAMP or cGMP
could antagonize the action of extracellular second
messenger (20). Elalamy and colleagues have provid-
ed compelling evidence for the involvement of an
ecto-PKA (protein kinase A) in mediating the actions
of extracellular cAMP (used at a concentration of 5
M) on expression of prostaglandin H synthase
(PGHS-2) in a pulmonary microvascular endothelial
REVIEWS
FIGURE 2. Blockade of cAMP extrusion with probenecid alters
intracellular free [cAMP]
Intracellular cAMP was imaged using a FRET-based biosensor (46) in sin-
gle HEK293 cells as described previously (25). This sensor (courtesy of Dr.
Kees Jalink) is based on the cAMP binding protein Epac, which has been
labeled with CFP and YFP. cAMP-dependent conformational changes of
the Epac protein result in changes in FRET, providing a measure of free
[cAMP]. Acute treatment of cells with 1 mM probenecid caused a small
but significant change in the resting FRET signal (the 480:535 nm emis-
sion ratio), consistent with an increase in intracellular [cAMP]. These data
suggest that the cAMP export process can contribute to intracellular
cAMP homeostasis, in addition to mediating the elevation in extracellular
cAMP. Shown for comparison is the action of the direct adenylyl cyclase
activator forskolin (50 M).
Extracellular Calcium as a Third
Messenger
Just as it has long been known that intracellular cAMP
signaling events are associated with extracellular
accumulation of the second messenger, so has it been
long appreciated that Ca
2+
can fluctuate outside cells,
owing to activation of influx and efflux pathways for
the cation during Ca
2+
signaling events (41). As with
cAMP, early measurements of hormone-stimulated
Ca
2+
signals frequently relied on determinations of
Ca
2+
in the external media. Because diffusion is great-
ly limited in the interstitial spaces (which occupy only
a fraction of the tissue volume; e.g., ~20% in brain
tissue) (66) and the buffering capacity for Ca
2+
inside
the cell is so much greater than outside, these fluxes
can lead to significant alterations in free [Ca
2+
] in the
extracellular milieu.
Fluctuations in Extracellular Ca
2+
As indicated in FIGURE 3, agonist-stimulated Ca
2+
signaling events involve 1) the release of Ca
2+
from
internal storage compartments into the cytoplasm
via intracellular release channels (i.e., the InsP
3
receptor); 2) the extrusion of Ca
2+
into the extracellu-
lar space by plasma membrane Ca
2+
ATPases (PMCA)
or other export mechanisms (e.g., Na
+
/Ca
2+
exchang-
ers); and 3) the activation of Ca
2+
entry through
store-operated channels (SOCs), such as the recently
identified Ca
2+
release-activated pathways known as
the Orai proteins (47).
Tepikin and colleagues have provided direct
demonstrations of the significant quantitative impact
of the Ca
2+
extrusion process on extracellular Ca
2+
levels adjacent to stimulated cells (61–63). One study
employed simultaneous real-time measurements of
intracellular and extracellular [Ca
2+
] in single pancre-
atic acinar cells suspended in a small droplet (40–90
times the volume of the cell; FIGURE 4). By measuring
the extracellular [Ca
2+
] in the droplet with a Ca
2+
-sen-
sitive dye, it was estimated that the total intracellular
calcium content was reduced by 0.7 mM during
cholinergic stimulation, owing to active transport of
the cation by the PMCA.
Temporal and spatial separation of Ca
2+
entry and
efflux across the plasma membrane can give rise to
physiologically significant excursions in extracellular
[Ca
2+
] (13, 35), particularly in polarized epithelial cells
and other functionally polarized cells such as neurons
(33). For example, Caroppo and colleagues showed
that [Ca
2+
] in the luminal micro-compartment of the
intact gastric gland increases by 200–500 M following
cholinergic stimulation, owing to an abundance of
PMCA on the apical membrane of the gastric
epithelial cells (13). At the same time, a comparable
depletion of Ca
2+
was recorded in the basolateral inter-
stitium of the intact mucosa as a result of Ca
2+
influx
via pathways located predominantly at the basal cell
side. As described below, these extracellular [Ca
2+
]
fluctuations have been shown to have functional con-
sequences.
It is also well established that specific elements of the
Ca
2+
-handling machinery (as well as certain Ca
2+
sen-
sors) can be confined in cell surface microdomains,
such as caveolae (17, 26), potentially giving rise to local
gradients of Ca
2+
in the caveolar nanospaces. Other fac-
tors can influence free [Ca
2+
] in the external milieu,
including dilution and concentration of ionic species,
owing to cellular water transport. In addition, the
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PHYSIOLOGY • Volume 22 • October 2007 • www.physiologyonline.org
REVIEWS
FIGURE 3. “Third messenger” activity of extracellular Ca
2+
Local extracellular [Ca
2+
] can fluctuate as a consequence of agonist-
induced intracellular Ca
2+
signaling events. In the typical scenario, acti-
vation of a G
q/11
-coupled receptor by a Ca
2+
-mobilizing agonist results
in inositol 1,4,5-trisphosphate (InsP
3
) production, giving rise to the lib-
eration of stored Ca
2+
via InsP
3
receptor release channels in intracellular
Ca
2+
pools. A substantial fraction of Ca
2+
released into the cytoplasm is
rapidly extruded by plasma membrane Ca
2+
ATPases (PMCAs), poten-
tially resulting in significant local elevation of the extracellular [Ca
2+
].
Store emptying also triggers Ca
2+
influx via store-operated Ca
2+
chan-
nels (SOCs) in the plasma membrane, leading to transient depletion of
Ca
2+
in the volume-limited interstitial spaces. The ensuing local fluctua-
tions in Ca
2+
can influence a variety of Ca
2+
-sensing proteins on adja-
cent cells or on the same cell. Examples include HERG K
+
channels,
several types of nonselective cation channels, and a host of G-protein-
coupled receptors modulated by extracellular Ca
2+
(e.g., the extracellu-
lar Ca
2+
-sensing receptor, the GPRC6A orphan receptor, or
metabotropic glutamtate receptors). Gap-junction hemichannels and
the transmembrane protein notch are also susceptible to alterations in
extracellular [Ca
2+
] (32).
Extracellular Ca
2+
Sensors
Although specific GPCRs for cAMP have not been
identified in mammalian cells, cell-surface receptors
for Ca
2+
are known to exist, and some of these have
been well characterized. Without question, the best
known of these is the extracellular calcium-sensing
receptor (CaR), which was originally cloned from
bovine parathyroid gland in 1993 by Brown and col-
leagues (9). The structural and functional properties
of this widely expressed divalent cation receptor have
been reviewed extensively elsewhere (10, 33) and will
not be addressed in detail here. The CaR (of which
only a single isoform appears to exist) is indispensa-
ble for life in mammals, acting as the Ca
2+
sensor that
controls systemic Ca
2+
and Mg
2+
homeostasis via PTH
secretion. An emerging literature describes numer-
ous physiological functions of this receptor through-
out the body and in diverse vertebrate species,
including birds and fish. Deletion of CaR is lethal, but
the developments of viable “rescued” CaR knockout
mouse models that maintain normal parathyroid
function are being used increasingly to examine this
receptor’s physiological role in other organ systems
(1, 45).
CaR is a member of family C of the GPCR superfam-
ily, which also includes three taste receptors (T1–T3),
the GABA
B
receptors, eight metabotropic glutamate
receptors (mGluR1–mGluR8), and six orphan recep-
tors, including the recently characterized GPRC6A (7,
68). These receptors appear to share an evolutionary
thread with CaR based on their common functional
origins as nutrient/salinity sensors (15, 30). The CaR is
allosterically modulated by extracellular amino acids
(15). Conversely, other members of this family that are
regarded as amino acid sensors, such as certain
mGluRs, GABA
B
receptors, and GPRC6A, are modulat-
ed by extracellular Ca
2+
(44). GPRC6A has 34% amino
acid sequence identity with CaR (68) and is activated
by relatively high extracellular [Ca
2+
] (5–10 mM) (44).
This receptor has been suggested to serve as a sensor
for Ca
2+
in bone (44), a tissue where local extremes in
external [Ca
2+
] are believed to occur during the bone
remodeling process.
Many other cell surface proteins are susceptible to
physiological fluctuations in external [Ca
2+
] (recently
reviewed in Ref. 32). These include gap-junction
hemichannels (64), which can open in response to a
modest (~200 M) decrease in external [Ca
2+
], and the
receptor Notch, which may sense external [Ca
2+
] to
drive the establishment of right-left symmetry during
embryogenesis (50, 51). A distinct Ca
2+
-sensing recep-
tor known as CAS has been recently described in
plants (59). In addition, a number of ion channels alter
their open probability depending on the local extracel-
lular Ca
2+
, including the proton-gated cation channels
ASIC1a/ASIC1b, HERG K
+
channels, and other nonse-
lective channels found in neuronal tissue (32).
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PHYSIOLOGY • Volume 22 • October 2007 • www.physiologyonline.org
transport of Ca
2+
buffers (e.g., HCO
3–
, PO
4
2–
) would also
be expected to influence the free [Ca
2+
] in the intersti-
tium. Ca
2+
taken up into endocytic vesicles could con-
ceivably impact the local extracellular [Ca
2+
] (23).
Finally, secretory vesicles are known to contain high
concentrations of Ca
2+
and other divalent cations (Zn
2+
,
Mg
2+
), and synchronous secretory activity could in
principle lead to rapid increases in extracellular diva-
lents (29, 48). Gray et al. recently proposed that libera-
tion of these metals from vesicles of insulin-secreting
cells may constitute a means of communication
between cells (29) via sensors for extracellular Ca
2+
, as
described in the following section.
REVIEWS
FIGURE 4. Extracellular [Ca
2+
] becomes elevated adjacent to
stimulated cells due to Ca
2+
export
Direct measurements of Ca
2+
extrusion from pancreatic acinar cells per-
formed by Tepikin and colleagues (62) using the “droplet technique”
demonstrates that large quantities of Ca
2+
are exported from stimulated
cells. The total drop in cellular Ca
2+
following acetylcholine (ACh) treat-
ment was estimated to be about 0.7 mM over 2–5 min (modified with
permission from Ref. 62). A: photomicrograph of fluid micro-droplet con-
taining a single mouse pancreatic acinar cell. The cell was loaded with the
Ca
2+
indicator fura-2, whereas the droplet contained a second Ca
2+
dye,
fluo-3. At right is seen the pipette tip, used for iontophoretic delivery of
agonists. B: simultaneous measurement of free intracellular Ca
2+
concen-
tration ([Ca
2+
]
i
) in a single acinar cell and extracellular Ca
2+
([Ca
2+
]
o
) in the
droplet following challenge with 20 nM ACh.
Intercellular Communication
Via Ca
2+
Our laboratory demonstrated some years ago in a
proof-of-concept study using a co-culture model sys-
tem that it is possible for CaR to detect extracellular
fluctuations in [Ca
2+
] that occur secondary to intracel-
lular Ca
2+
signaling events (34, 65). This opened up the
prospect that Ca
2+
might function as a paracrine
messenger, used, for example, to communicate infor-
mation about the signaling status of a neighboring cell
or to integrate or reinforce signals in multicellular
ensembles. We further provided evidence for a varia-
tion on this theme, whereby exported Ca
2+
can activate
CaR expressed on the same cell in an autocrine fash-
ion (19). Caroppo et al. (13) later showed a physiolog-
ical role of this third messenger signaling system in the
intact gastric mucosa. These investigators took advan-
tage of information gained from their previous extra-
cellular microelectrode studies aimed at measuring
the profile of the extracellular Ca
2+
“signal” in the
apical and basolateral microdomains following
cholinergic stimulation (see above) (13). Remarkably,
reproducing this physiological pattern of extracellular
[Ca
2+
] variation was able to elicit changes in pepsino-
gen and alkaline secretion from the tissue (14), and
more recently this third messenger activity has been
linked to changes in water transport (24) in the same
model system. The CaR, which is expressed apically in
the amphibian oxyntic cell, is involved in detecting the
extracellular [Ca
2+
] elevation that occurs in the lumi-
nal compartment of the gastric gland, although it
appears that another entity may be responsible for
sensing the basolateral decrease in [Ca
2+
] (14). These
intriguing data are suggestive of a novel mode of Ca
2+
signaling that takes advantage of extracellular, rather
than intracellular, changes in [Ca
2+
], but it remains to
be seen whether this process occurs in other tissues.
Other Second Messengers as “Third
Messengers”?
Are there other hydrophilic signaling molecules that
are exported by cells to inform neighboring cells of
their signaling or metabolic status? Cyclic GMP, the
second messenger generated by either atrial natri-
uretic peptide or nitric oxide gas via guanylate
cyclases, is vigorously exported from many cell types
in quantities that surpass that of cAMP (4, 54). This
widespread phenomenon is mediated by many of the
same MRP family members (e.g., MRP4, MRP5,
MRP8) known to transport cAMP, as well as the
organic anion transporter OAT1 (69). Diverse biologi-
cal actions of extracellular cGMP have been described
in brain and kidney [recently reviewed by Sager (54)],
but as is the case for extracellular cAMP, specific
molecular receptors for cGMP in mammalian cells
have yet to be identified.
Isolated reports of extracellular accumulation of
inositol 1,4,5 trisphosphate (InsP
3
) following choliner-
gic stimulation as measured using microdialysis tech-
niques in brain have also appeared (40, 52). Roberts et
al. found that several inositol phosphate metabolites
of InsP
3
appeared (in addition to InsP
3
) in the intersti-
tial space under these conditions, although it is uncer-
tain whether the appearance of the additional inositol
derivatives reflects metabolism of extracellular InsP
3
or a separate transport process (52). It is not known
whether InsP
3
egress is a widespread phenomenon or
whether it has any functional significance.
Conclusions
Second messengers are the cellular currency of infor-
mation transfer. However, the generation of cAMP from
ATP and the energy required to maintain the gradients
that permit Ca
2+
signaling to take place come at a cer-
tain energetic cost. Thus it is attractive to imagine that
multicellular organisms might capitalize on fluctua-
tions in extracellular second messengers to expand the
informational content of the intracellular signal trans-
duction process. The concept of the interstitial
microdomain as a specialized signaling compartment
is in its infancy, however. There is still much to learn
about how and when the local concentrations of cAMP
and Ca
2+
change in this space and what the physiologi-
cal consequences of these fluctuations are. The devel-
opment of practical methods for probing the profile of
such extracellular “signals” will be an important first
step to understanding whether this constitutes a gener-
alized device to extend the scope and range of second
messenger molecules to a domain outside the cell.
We are grateful to Dr. Kees Jalink of the Netherlands
Cancer Institute for graciously providing us with the Epac-
based FRET sensor used for imaging intracellular cAMP
and to our colleague Prof. Silvana Curci for reading the
manuscript.
Support for the work conducted in the Hofer laboratory
was provided by the Medical Research Service of the
Department of Veteran’s Affairs and the Brigham Surgical
Group.
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